九疑山兔线粒体基因组组装及系统进化分析

doi:10.11843/j.issn.0366-6964.2024.10.015

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4417-4427.doi: 10.11843/j.issn.0366-6964.2024.10.015

九疑山兔线粒体基因组组装及系统进化分析

李聪聪¹(), 黄子珂¹, 黄念旎¹, 马诗语¹, 刘晗¹, 肖志标², 宋果², 蒋亮², 彭为波², 杨联熙³, 郭云涛⁴, 黄生强¹^,*()

1. 湖南农业大学动物科学技术学院, 长沙 410128
2. 宁远县畜牧水产事务中心, 永州 425600
3. 湖南伊普吕农牧科技有限公司, 永州 425600
4. 中科基因生物科技(江苏)有限公司, 南通 226000

收稿日期:2024-03-07 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 黄生强 E-mail:lcc199308@126.com;hsq07@126.com
作者简介:李聪聪(1993-), 女, 河南南阳人, 硕士, 主要从事家兔遗传与分子育种研究, E-mail: lcc199308@126.com
基金资助:
湖南省科技厅项目：九疑山兔选优提纯研究(2019NK4192)

Mitochondrial Genome Assembly and Phylogenetic Analysis of the Jiuyishan Rabbit

Congcong LI¹(), Zike HUANG¹, Nianni HUANG¹, Shiyu MA¹, Han LIU¹, Zhibiao XIAO², Guo SONG², Liang JIANG², Weibo PENG², Lianxi YANG³, Yuntao GUO⁴, Shengqiang HUANG¹^,*()

1. College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China
2. Animal Husbandry and Fisheries Affairs Center of Ningyuan County, Yongzhou 425600, China
3. Hunan Hyplus Agricultural and Pastoral Technology Co., Ltd., Yongzhou 425600, China
4. The Medical Laboratory of Nantong ZhongKe Co., Ltd., Nantong 226000, China

Received:2024-03-07 Online:2024-10-23 Published:2024-11-04
Contact: Shengqiang HUANG E-mail:lcc199308@126.com;hsq07@126.com

摘要/Abstract

摘要：

旨在对九疑山兔线粒体基因组进行组装，探究其系统进化和分类地位。本研究采用高通量测序技术对20只150日龄九疑山兔的耳组织进行了全基因组测序，利用生物信息学软件对线粒体基因组进行组装，并对获得的线粒体基因组进行序列分析、基因预测和注释，随后基于已发表的家兔、野兔、经济动物和模式动物的线粒体基因组序列进行多序列比对和系统进化分析，对研究群体的多态性位点进行鉴定。组装获得长度为17 306 bp的九疑山兔线粒体基因组全序列，其碱基组成、基因分布与已发表的5个家兔品种的线粒体基因组基本一致。基于D-loop区的系统进化分析表明, 九疑山兔与福建黄兔进化关系较近，而与欧洲穴兔、沂蒙毛兔、川白獭兔和新西兰白兔较远。通过其与14种野兔及11种经济/模式动物的种间系统进化分析表明, 九疑山兔与欧洲野兔、海南兔和白靴兔亲缘关系较近，与其他野兔和经济/模式动物较远。研究群体的线粒体基因组保守区共鉴定到了12个突变位点，其中有3个突变的群体等位基因频率大于0.8，为多态性位点。本研究组装获得了首个九疑山兔的完整线粒体基因组序列，明确了九疑山兔与其它品种家兔、野兔和常见经济/模式动物的亲缘关系和分类地位，为九疑山兔的种质资源利用和品种选育等研究提供了基础数据。

关键词: 九疑山兔, 线粒体基因组, 高通量测序, 系统进化

Abstract:

The aim of the study was to assemble the mitochondrial genome of the Jiuyishan rabbit and to investigate its phylogenetic evolution and taxonomic position. High-throughput sequencing technology was employed to perform whole-genome sequencing on ear tissues from 20 Jiuyishan rabbits at 150 days of age. Bioinformatics software was employed for the assembly of the mitochondrial genome, followed by conducting sequence analysis, gene prediction and annotation of the obtained mitochondrial genome. Subsequently, multiple sequence alignment and phylogenetic analysis were conducted based on the published mitochondrial genome sequences of rabbits, hares, economically important animals and model animals. Polymorphic sites within the study population were identified. The complete mitochondrial genome sequence of the Jiuyishan rabbit was successfully obtained, the length was 17 306 bp. The nucleotide composition and gene arrangement of the mitochondrial genome were found to be largely consistent with those reported for 5 previously published domestic rabbit breeds. Phylogenetic analysis based on the D-loop region indicated a closer evolutionary relationship between the Jiuyishan rabbit and the Fujian Yellow rabbit, while Jiuyishan rabbit was more distantly related to the European rabbit, Yimeng Wool rabbit, Chuanbai Rex rabbit and New Zealand White rabbit. Interspecific phylogenetic analysis involving 14 wild rabbit species and 11 economic or model animal species demonstrated that the Jiuyishan rabbit shared a closer phylogenetic relationship with the European wild rabbit, Hainan rabbit, and the White-booted rabbit but was further with others. A total of 12 mutation sites were identified in the conserved regions of the mitochondrial genome of the study population, with 3 mutations having allele frequencies greater than 0.8, indicating polymorphism. This study has successfully assembled the first complete mitochondrial genome sequence of the Jiuyishan rabbit, clarifying its phylogenetic relationships and taxonomic status in comparison to other domestic rabbit breeds, wild rabbits and common economic or model animals. This work provides foundational data for the utilization of genetic resources and the breeding of the Jiuyishan rabbit.

Key words: Jiuyishan rabbit, mitochondrial genome, high-throughput sequencing, phylogenetic evolution

中图分类号:

S829.1

李聪聪, 黄子珂, 黄念旎, 马诗语, 刘晗, 肖志标, 宋果, 蒋亮, 彭为波, 杨联熙, 郭云涛, 黄生强. 九疑山兔线粒体基因组组装及系统进化分析[J]. 畜牧兽医学报, 2024, 55(10): 4417-4427.

Congcong LI, Zike HUANG, Nianni HUANG, Shiyu MA, Han LIU, Zhibiao XIAO, Guo SONG, Liang JIANG, Weibo PENG, Lianxi YANG, Yuntao GUO, Shengqiang HUANG. Mitochondrial Genome Assembly and Phylogenetic Analysis of the Jiuyishan Rabbit[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4417-4427.

图/表 7

表 1

表 2

图 1

表 3

九疑山兔线粒体基因组结构"

基因 Gene	开始 Start	结束 End	长度/bp Length	链 Strand	A含量/% Per_A	T含量/% Per_T	C含量/% Per_C	G含量/% Per_G	AT含量/% Per_AT	CG含量/% Per_CG
tRNA-Phe	1	69	69	H	37.68	21.74	23.19	17.39	59.42	40.58
rrnS	70	1 026	957	H	35.84	23.93	22.05	18.18	59.77	40.23
tRNA-Val	1 027	1 092	66	H	30.30	27.27	25.76	16.67	57.58	42.42
rrnL	1 093	2 671	1 579	H	36.35	25.46	20.39	17.80	61.81	38.19
tRNA-Leu	2 672	2 746	75	H	32.00	26.67	22.67	18.67	58.67	41.33
ND1	2 749	3 705	957	H	28.74	28.84	30.09	12.33	57.58	42.42
tRNA-Ile	3 704	3 773	70	H	37.14	34.29	11.43	17.14	71.43	28.57
tRNA-Gln	3 771	3 842	72	L	31.94	25.00	29.17	13.89	56.94	43.06
tRNA-Met	3 851	3 919	69	H	28.99	27.54	24.64	18.84	56.52	43.48
ND2	3 920	4 963	1 044	H	34.96	28.26	27.68	9.10	63.22	36.78
tRNA-Trp	4 971	5 037	67	H	34.33	22.39	23.88	19.40	56.72	43.28
tRNA-Ala	5 040	5 106	67	L	32.84	29.85	22.39	14.93	62.69	37.31
tRNA-Asn	5 107	5 179	73	L	28.77	24.66	28.77	17.81	53.42	46.58
tRNA-Cys	5 212	5 280	69	L	24.64	28.99	24.64	21.74	53.62	46.38
tRNA-Tyr	5 281	5 346	66	L	27.27	33.33	22.73	16.67	60.61	39.39
COX1	5 354	6 895	1 542	H	26.39	31.06	25.23	17.32	57.46	42.54
tRNA-Ser	6 898	6 966	69	L	33.33	21.74	27.54	17.39	55.07	44.93
tRNA-Asp	6 970	7 038	69	H	36.20	39.13	14.49	10.14	75.36	24.64
COX2	7 039	7 722	684	H	30.26	28.65	26.61	14.47	58.92	41.08
tRNA-Lys	7 726	7 794	69	H	31.88	26.09	18.84	23.19	57.97	42.03
ATP8	7 796	7 999	204	H	34.31	28.43	29.41	7.84	62.75	37.25
ATP6	7 957	8 637	681	H	29.52	29.66	29.52	11.31	59.18	40.82
COX3	8 637	9 419	783	H	27.20	28.74	28.74	15.33	55.94	44.06
tRNA-Gly	9 421	9 490	70	H	32.86	30.00	22.86	14.29	62.86	37.14
ND3	9 491	9 847	357	H	31.37	33.05	23.81	11.76	64.43	35.57
tRNA-Arg	9 838	9 904	67	H	40.30	35.82	10.45	13.43	76.12	23.88
ND4L	9 906	10 202	297	H	29.63	34.34	25.59	10.44	63.97	36.03
ND4	10 196	11 572	1 377	H	30.14	29.27	29.19	11.40	59.40	40.60
tRNA-His	11 574	11 642	69	H	37.68	30.43	17.39	14.49	68.12	31.88
tRNA-Ser	11 643	11 701	59	H	25.42	28.81	28.81	16.95	54.24	45.76
tRNA-Leu	11 702	11 771	70	H	41.43	25.71	14.29	18.57	67.14	32.86
ND5	11 772	13 583	1 812	H	30.85	30.63	27.04	11.48	61.48	38.52
ND6	13 579	14 103	525	L	40.38	19.81	33.33	6.48	60.19	39.81
tRNA-Glu	14 104	14 171	68	L	36.76	30.88	17.65	14.71	67.65	32.35
CYTB	14 175	15 314	1 140	H	27.98	28.95	30.35	12.72	56.93	43.07
tRNA-Thr	15 314	15 379	66	H	28.79	27.27	21.21	22.73	56.06	43.94
tRNA-Pro	15 380	15 445	66	L	28.79	21.21	34.85	15.15	50.00	50.00
D-loop	15 446	17 306	1 861	H	31.06	24.61	30.63	13.70	55.67	44.33

表 3

图 2

图 3

表 4

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分类 Classification	拉丁名 Latin name	中文名 Chinese name	GenBank登录号 GenBank accession No.
家兔Rabbit	Oryctolagus cuniculus	新西兰白兔	MH985853.1
	Oryctolagus cuniculus	川白獭兔	MN953621.1
	Oryctolagus cuniculus	沂蒙毛兔	MN296708.1
	Oryctolagus cuniculus	欧洲穴兔	NC_001913.1
	Oryctolagus cuniculus	福建黄兔	MN518689.1
野兔Hare	Lepus oiostolus	高原兔	NC_050983.1
	Lepus yarkandensis	塔里木兔	NC_050569.1
	Lepus hainanus	海南兔	NC_025902.1
	Lepus europaeus	欧洲野兔	NC_004028.1
	Lepus americanus	白靴兔	NC_024043.1
	Lepus timidus	雪兔	NC_024040.1
	Lepus capensis	草兔-开普野兔	NC_015841.1
	Lepus tibetanus pamirensis	藏兔	LC073697.1
	Lepus sinensis	华南兔	NC_025316.1
	Lepus tolai	草兔-托氏兔	NC_025748.1
	Lepus coreanus	韩国野兔	NC_024259.1
	Lepus granatensis	格拉纳达野兔	NC_024042.1
	Lepus townsendii	白尾长耳大野兔	NC_024041.1
	Lepus arcticus	北极兔	NC_044769.1
外群External species	Ovis aries	绵羊	NC_001941.1
	Capricornis crispus	山羊	NC_005044.2
	Homo sapiens	人	NC_012920.1
	Bos taurus	牛	NC_006853.1
	Equus caballus	马	NC_001640.1
	Sus scrofa	猪	NC_012095.1
	Gallus gallus	鸡	NC_040902.1
	Danio rerio	斑马鱼	NC_002333.2
	Drosophila immigrans	果蝇	NC_044669.1
	Mus musculus	小鼠	NC_005089.1
	Rattus norvegicus	大鼠	NC_001665.2

样本编号 Sample ID	总数据量/G Total data amount	Q30/%	GC含量/% GC content	插入片段长度/bp Insert size	比对率/% Mapping rate	平均深度(×) Average depth	线粒体平均深度(×) Average depth of the mitochondria genome
S1	58.83	93.79	45.20	290	99.02	18.83	3 741.22
S2	52.51	93.06	43.87	324	99.15	17.32	3 110.19
S3	53.71	92.77	43.67	343	99.11	18.15	5 757.16
S4	52.11	92.72	43.13	324	99.21	17.35	6 505.76
S5	53.98	92.86	43.12	334	99.28	18.33	10 553.55
S6	51.34	93.09	43.97	334	99.08	17.34	9 259.27
S7	52.34	93.35	43.72	321	99.14	17.32	4 583.41
S8	53.22	93.27	43.34	326	99.18	17.85	5 490.54
S9	51.09	89.87	42.61	374	99.21	17.71	5 217.01
S10	54.63	92.57	43.21	349	99.11	18.69	8 937.94
S11	55.35	93.06	43.32	327	99.21	18.53	6 778.39
S12	58.89	93.26	43.44	337	99.12	19.93	8 297.15
S13	52.44	93.81	42.75	356	99.17	18.03	5 308.23
S14	52.72	94.20	42.99	332	99.20	17.84	5 193.53
S15	58.10	94.03	43.38	331	99.17	19.68	2 968.24
S16	56.20	93.55	43.20	348	99.17	19.13	3 254.84
S17	56.34	93.27	43.14	339	99.21	19.12	3 918.06
S18	60.49	93.33	43.16	349	99.16	20.65	3 944.40
S19	48.38	93.09	43.70	364	99.12	16.63	2 805.76
S20	53.52	93.79	43.16	341	99.18	18.18	3 922.48
Mean	54.31	93.14	43.40	337	99.16	18.33	5 477.36
Min	48.38	89.87	42.61	290	99.02	16.63	2 805.76
Max	60.49	94.20	45.20	374	99.28	20.65	10 553.55

线粒体位置 Location	参考碱基 Reference base	突变碱基 Alternative base	突变功能 Mutation function	突变类型 Mutation type	突变所在基因名 Gene name	HGVS写法(外显子) HGVS.c	HGVS写法(蛋白) HGVS.p	杂合个体数 Heterozygote number	纯合个体数 Homozygote number	群体等位基因频率 MAF	携带突变个体 Rabbits carrying mutations
1647	A	T	ncRN$\underline{\rm{A}}$ exonic	.	rrnaL	.	.	0	1	0.05	S12
2228	T	C	ncRN$\underline{\rm{A}}$ exonic	.	rrnaL	.	.	0	3	0.15	S4/S6/S10
4643	T	G	exonic	missens$\underline{\rm{e}}$ variant	ND2	c.724T>G	p.Ser242Ala	0	1	0.05	S12
5195	-	A	intergentic	.	.	.	.	2	0	0.05	S15/S18
5870	G	C	exonic	missens$\underline{\rm{e}}$ variant	COX1	c.517G>C	p.Ala173Pro	0	20	1	S1/S2/S3/S4/S5/ S6/S7/S8/S9/S10/ S11/S12/S13/S14/ S15/S16/S17/S18/ S19/S20
6781	C	T	exonic	synonymou$\underline{\rm{s}}$ variant	COX1	c.1428C>T	p.Phe476Phe	0	6	0.3	S13/S14/S16/ S17/S19/S20
7379	A	G	exonic	missens$\underline{\rm{e}}$ variant	COX2	c.341A>G	p.Glu114Gly	0	2	0.1	S16/S20
10402	G	A	exonic	synonymou$\underline{\rm{s}}$ variant	ND4	c.207G>A	p.Thr69Thr	0	1	0.05	S3
10894	T	C	exonic	synonymou$\underline{\rm{s}}$ variant	ND4	c.699T>C	p.Ala233Ala	0	2	0.1	S5/S9
11565	T	C	exonic	missens$\underline{\rm{e}}$ variant	ND4	c.1370T>C	p.Leu457Pro	0	19	0.95	S2/S3/S4/S5/S6/ S7/S8/S9/S10/ S11/S12/S13/S14/ S15/S16/S17/S18/ S19/S20
12400	T	C	exonic	missens$\underline{\rm{e}}$ variant	ND5	c.629T>C	p.Ile210Thr	0	1	0.05	S12
15341	T	C	ncRN$\underline{\rm{A}}$ exonic	.	tRNA-Thr	.	.	0	17	0.85	S1/S2/S3/S4/S6/ S7/S8/S10/S11/ S13/S14/S15/ S16/S17/S18/ S19/S20

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九疑山兔线粒体基因组组装及系统进化分析

Mitochondrial Genome Assembly and Phylogenetic Analysis of the Jiuyishan Rabbit

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